1. Field of the Invention
The present invention concerns a method of inducing the formation of adventitious buds, adventitious embryos or bulblets of a plant by applying tissue culture to plants in a specific method.
2. Description of the Prior Art
Vegetables, such as cabbages, tomatoes and cucumbers as well as rices have been utilized for foods, while on the other hand, horticultural plants such as tulips, bluebottle and rudbeckia have been favored as ornamental plants. These plants have been multiplied traditionally by seedlings, division of bulbs or tubers, etc. However, such multiplying methods not only require a large area and much labor, they also involve problems of reduction of seedling growing rate or degradation in the quality of flowers due to the spread of virus diseases in recent years. To maintain and promote superior properties of plants, a vegetative propagation may be suitable. With the aim of avoiding these problems, improving the overall process and increasing multiplication efficiency, methods utilizing the techniques of plant tissue culture have also been reported in recent years (for example, refer to Japanese Patent Laid-Open No. Sho 55-14734). Multiplication by tissue culture techniques has been attained by way of differentiation of adventitious buds, adventitious embryos, bulbs, etc. from cultured tissue pieces and cultured cells and it has been considered that the differentiation is controlled by the concentration ratio between cytokinin and auxin as a plant hormone (for example, refer to Annals of Botany vol. 45, 321-327, 1980). However, there are many kinds of plant groups which cause no differentiation by plant hormones only and the frequency of differentiation therein, if it occurs, is extremely low. A more direct and effective method of inducing the differentiation is still needed.
The present inventors have investigated more efficiently multiplying seedlings of plants as compared with the usual case based on the recognition that there are various problems in plant tissue culture techniques as described above.
As a result, the present inventors have found those substances for promoting the differentiation of adventitious buds, adventitious embryos and bulbs of the plants acting on the plant cells and then found a method of multiplying the seedlings of plants efficiently based on these findings. That is, the method according to the present invention provides a method of inducing formation of adventitious buds, adventitious embryos or bulblets of a plant and a way of multiplying seedlings of plants by applying tissue culture after introducing calmodulin and/or calcium into the cells of plant tissue pieces or callus of the plants.
The plants that can be treated with the method of tissue culture according to the present invention are not restricted so the method can be applied to all kinds of plants.
Those plants to which the method according to the present invention can be applied include, for example, Papaveraceae, Solanaceae, Umbelliferae, Rosaceae, Liliaceae, Compositae, Geraniaceae, Cucurbitaceae and Gramineae. Specific examples of such plants include those described in "Ground for the plants System Classification" edited by Yamagishi, published by Kokuryukan in 1974. More specifically, there can be mentioned those plants belonging to the family Solanaceae such as eggplant, tomato, potato, sweet potato and basil; plants belonging to Papaveraceae such as poppy, rape, cabbage, radish, Chinese cabbage; plants belonging to Umbelliferae such as carrot, Japanese parsley and parsley, plants belonging to Rosaceae such as rose, strawberry, soybean and cherry; plants belonging to Liliaceae except for Lilium such as onions and tulips, plants belonging to Compositae such as chrysanthemum, bluebottle and sunflower; plants belonging to Geraniaceae such as pelargonium, geranium and flax, plants belonging to Cucurbitaceae such as cucumber and pumpkin; and plants belonging to Gramineae such as rice and corn. Among these plants relevant to the present invention, preferred plants are, specifically, tomato, tobacco, eggplant, trenia, carrot, cabbage, onion, soybean, basil, bluebottle, rudbeckia, carnation, trumpet lily, tulip, asparagus, flax, cucumber and rice.
In the present invention, the tissue culture of plants can be conducted by using tissue pieces or callus of the plant. The tissue culture pieces can include, specifically, tissue pieces of plants prepared by slicing cotyledon, hypocotyl, shoot apex, stalk, leaf, scale or root of other tissue. These tissue pieces are used usually after sterilization with sodium hypochlorite or ethanol. However, in the case of using an aseptically cultured plant, the above-mentioned sterilized procedure is not required. Further, in the case of multiplying seedlings of plants with no diseases and viruses, tissues near the apical point and tissue pieces of the plants obtained from the tissue near the apical point can be used as the culture material. The cultured cells that can be used in the tissue culture of the plants in the present invention are obtained from any available portion of the plant and are non-differentiated amorphous cells including callus tissue obtained by applying tissue culture to the tissue pieces by the known method.
In the present invention, for forming a seedling of a plant by the tissue culture of tissue pieces or cultured cells of the plant thereby forming the seedling, the method as specifically described below is used.
In the method of applying tissue culture according to the present invention, tissue culture is applied after introducing calmodulin and/or calcium into the cells of the tissue pieces or callus.
Calmodulin used in the present invention is a sort of protein such as Ca.sup.2+ -ATPase of an erythrocyte membrane or phosphodiesterase of the brain having an activating function, the mobility of which is reduced in the presence of calcium upon conducting electrophoresis. Calmodulin can be isolated and purified for example, from bulbs of trumpet Lily, cerebrum of cattle, seminal vesicle of rat, etc.
As a specific illustration of the present invention of applying tissue culture after introducing calmodulin and/or calcium into the cells is now described. (1) The tissue culture method according to the present invention provides a method of applying tissue culture after introducing a specific protein referred to as calmodulin into the cells of tissue pieces or callus of plants.
The method of introducing calmodulin according to the present invention can include, for example, the following methods. The calmodulin introducing treatment is carried out by placing tissue pieces or callus of a plant to be subjected to tissue culture between electrodes and, after adding a solution containing calmodulin thereto, electrical pulses at from 30 V/cm to 2.5 KV/cm of electrical field intensity are applied for 30 .mu.sec to 1 msec. In this case, calmodulin present in the solution is desirably used at a concentration usually from 3 .mu.g/ml to 1 mg/ml. In addition to the electrical injection process as described above, there can be mentioned various ways techniques for injecting a solution containing calmodulin into the inside of the cells by using a micropipet made of glass under microscopic observation, a method of irradiating laser beam pulses to the cells placed in a calmodulin-containing solution to punctuate fine pores of submicron diameter to the cells and introducing calmodulin through the holes to the inside of the cells by using a method of transporting the material to the inside of the living cells as described Japanese Patent Publication No. Sho 62-7838, or a method of introducing macromolecules to the inside of plant cells by using tungsten bullets reported recently, etc. The amount of calmodulin introduced to the inside of the cells by the methods described above in the present invention can be determined by labelling the calmodulin with a fluorescent dye and measuring the fluorescence intensity after introduction to the inside of the cells. The amount is within a range usually from 1 .mu.g/ml to 300 .mu.g/ml and, preferably, from 10 .mu.g/ml to 100 .mu.g/ml.
In the method of applying tissue culture after mechanically introducing calmodulin to the inside of the cells of tissue pieces or callus of plants according to the present invention, tissue culture may be applied using a culture medium prepared by adding calcium ions at a concentration usually greater than 1 mM to the medium described later, after the introduction of calmodulin. In addition, after the introduction of calmodulin, tissues or calluses may be cultured using a culture method such as proposed in U.S. patent application Ser. No. 07/162,520(Japanese Patent Laid-Open Application No. Sho 63-279,772) according to the present inventors. That is, tissue culture may be applied using a culture medium containing calcium ionophores such as A23187 within the range from 10.sup.-8 to 10.sup.-4 M, preferably from 10.sup.-7 to 10.sup.-5 M. In addition, after tissue culture was applied by using a culture medium containing calcium ionophores such as A23187 within the range from 10.sup.-8 to 10.sup.-4 M, the tissue pieces or callus of plants may be further cultured by mechanically adding calmodulin. (2) The tissue culture according to the present invention provides a method of applying tissue culture after introducing calcium ion into the cells of tissue pieces or the callus of plants.
The calcium introduction method according to the present invention can be conducted in the same manner as in the case of introducing calmodulin. For instance, in the case of electrical injection, the tissue pieces or callus of a plant are put between electrodes, an aqueous solution containing calcium ion is added and, thereafter, pulses are applied to introduce calcium ion to the inside of the cells of the tissue pieces or callus.
The calcium ion concentration in the solution in this case, is usually within a range from 100 .mu.M to 30 mM. The solution containing calcium ion can include, specifically, those aqueous solutions containing dissolved therein calcium compounds such as calcium chloride, calcium nitrate and calcium carbonate. In the present invention, the amount of calcium ion introduced to the inside of the cells by the above-mentioned method, when expressed in the same manner as for calmodulin, is within a range usually from 10.sup.-8 M to 10.sup.-5 M and, preferably, from 10.sup.-6 M to 10.sup.-5 M. (3) The tissue culture method of the present invention provides a method of applying tissue culture after introducing calmodulin and calcium into the cells of tissue pieces or cultured cells of plants.
The above-mentioned method is particularly preferred in the present invention since the differentiation for the adventitious buds and adventitious embryos of plants is remarkably promoted as compared with the methods (1), (2) above. The calmodulin and calcium ion can be introduced into the cells by the same procedures as in the case of (1) and (2) mentioned above and introduction can be attained, for instance, by placing the tissue pieces or callus of plants between electrodes and, after adding a solution containing calcium ion and calmodulin, applying pulses at the same electrical field intensity as above in the same manner. In this case, the concentrations for the calmodulin and calcium ion in the solution are within the same ranges as those in (1) and (2) above. Further, the amounts of calmodulin and calcium ion introduced to the inside of the cells are also within the range of (1) and (2) described above.
Upon introducing calmodulin and calcium ion to the inside of cells of the tissue pieces or callus in the present invention, it is usually desirable to simultaneously introduce calcium ion and calmodulin at the same time using a solution containing calcium ion and calmodulin as described above. It is also possible to separately introduce calmodulin and calcium, as required, to the inside of the cells by the method as described above, etc., followed by applying tissue culture.
Culture medium used for the tissue culture applied after the introduction of calmodulin and/or calcium can include, for example, those culture media as specifically described later.
The culture medium used in the present invention contains inorganic ingredients and a carbon source as the essential ingredients, to which plant hormones and vitamins and, as required, amino acids are added. The inorganic ingredients for the culture medium can include those inorganic salts including elements such as nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine and cobalt. Specifically, these compounds include potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid and cobalt chloride.
The carbon source for the culture medium can include, for example, carbon hydrate and derivatives thereof such as sucrose, organic acids such as fatty acid and primary alcohols such as ethanol.
Plant hormones for the culture medium can include, for example, auxins such as naphthalene acetic acid (NAA), indole acetic acid (IAA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxy acetic acid (2,4-D), indole butyric acid (IBA) and derivatives thereof, and cytokinins such as benzyl adenine (BA), kinetin, zeatin, etc.
Vitamins for the culture medium can include, for example, biotine, thiamine (vitamin B1), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantotate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinic amide and riboflavine (vitamin B2).
The amino acid for the culture medium can include, for example, glycine, alanine, glutamic acid, cystein, phenyl alanine and lysine.
The culture medium of the present invention desirably contains from about 0.1 .mu.M to about 100 mM of the inorganic ingredient, from about 1 g/l to about 100 g/l of a carbon source, from about 0.01 mg/l to about 10 mg/l of plant hormones, from about 0.1 mg/l to about 150 mg/l of vitamins and from 0 to about 1000 mg/l of amino acids.
The culture medium used for the tissue culture according to the present invention can include specifically those known culture media used for the tissue culture, for example, Murashige & Skoog culture medium ('62), Linsmaier & Skoog culture medium (RM-1965), White culture medium ('63), Gamborg B-5 culture medium, Mitsui M-9 culture medium, Nitch & Nitch culture medium, etc., such media being incorporated as required with the carbon source and plant hormone as described above and, further, vitamins and amino acids as described above. Among them, those culture media prepared by using Nitch & Nitch, Linsmaier & Skoog or Marushige-Skoog media are preferred. In the present invention, it is also possible to use a culture medium prepared by adding calcium ionophore, cyclic AMP and polyamine as proposed in Japanese Patent Laid-open Application No. Sho 631-2779722 mentioned above. The compositions of the known culture media mentioned above are described, for example, in "New Plant Tissue Culture" P386-P391, written by Takeuchi, Nakajima, Furuya, and published by Asakura Shoten in 1979.
The culture medium usable in the present invention is a liquid culture medium or solid culture medium usually containing from 0.1 to 2% of a gelling agent such as agar or Gelite.
In the present invention, the tissue pieces or callus of plants as described above can be subjected to tissue culture by using a liquid culture medium aerated with oxygen-containing gas in the same manner as described in Japanese Patent Laid-Open Application No. Sho 61-285928 of the present applicant.
According to the method of the present invention, it is possible to obtain a great number of adventitious buds, adventitious embryos, bulblets (small bulbs), etc. at high efficiency from tissue pieces or callus of plants. Adventitious buds obtained by the method according to the present invention can be rooted into plant bodies, which are then sliced into tissue pieces (bulblets are also sliced) and subjected further to the tissue culture by the culture method as described above according to the present invention thereby enabling one to multiply seedlings in a significant amount.
Further, the plants obtained by the present invention can be grown into intact, stable plant bodies by the usual cultivation.
Using the present invention, in which tissue culture is applied after introducing calmodulin and/or calcium into the cells of tissue pieces of or callus of plants, since the differentiation of adventitious buds, adventitious embryos and bulbs of plants is remarkably promoted, a great number of seedlings can be multiplied. Accordingly, by the method of the present invention, plant bodies of high quality can be cultured in a great number at higher efficiency as compared with the conventional method from the tissue pieces or callus of plants thereby enabling one to multiply seedlings in great number. Furthermore, by the use of the method according to the present invention, the grown stalks from the differentiated adventitious buds can have lager diameter stalks and one can obtain a good seedlings for that reason.